Nuclear Medicine and Biology 42 (2015) 171–176

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

In vivo radioactive metabolite analysis for individualized medicine:
A basic study of a new method of CYP activity assay using 123I-IMP
Kodai Nishi a,b, Asuka Mizutani b, Naoto Shikano c, Ken-ichi Fujita d, Masato Kobayashi b, Masahiro Ono b,e,
Ryuichi Nishii f, Yasutuna Sasaki d, Seigo Kinuya g, Keiichi Kawai b,h,⁎a

Department of Radioisotope Medicine, Atomic Bomb Disease institute, Nagasaki University, Nagasaki, Japan
School of Health Science, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan
Department of Radiological Sciences, Ibaraki Prefectural University of Health Science, Ibaraki, Japand
Institute of Molecular Oncology, Showa University, Tokyo, Japane
Research Center, Nihon Medi-Physics Co., Ltd., Chiba, Japanf
University of Miyazaki Hospital, Miyazaki, Japang
School of Medical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japanh
Biomedical Imaging Research Center, University of Fukui, Fukui, Japanbca r t i c l e

i n f oArticle history:
Received 20 May 2014
Received in revised form 25 August 2014
Accepted 25 August 2014
Keywords:
CYP activity
123
I-IMP
Individualized medicine
Metabolite analysis
Mousea b s t r a c t
Introduction: 123 I-N-isopropyl-p-iodoamphetamine ( 123 I-IMP) is metabolized and converted to 123 I-piodoamphetamine ( 123I-PIA) by CYP2C19 in humans. Since variations in 123I-PIA levels reﬂect variations
in CYP2C19 activity, CYP2C19 activity can be estimated by quantitative analysis of 123I-PIA levels. Thus, 123I-IMP
administration can provide diagnostic information not only regarding cerebral blood ﬂow (rCBF) but also
regarding metabolic function. The aim of the present study was to detect variations in CYP activity in miceusing metabolite analysis.
Methods: Metabolism of 125 I-IMP in pooled homogenates of mouse liver (MLH) was analyzed by highperformance liquid chromatography (HPLC) in the presence or absence of NADPH. The amount of 125I-PIA
generated was calculated as the normalized peak area of the chromatogram. Inhibition of 125I-IMP metabolism
was evaluated using the inhibitor SKF-525A. A biodistribution study of 125I-IMP was performed to determine theorgan distribution of 125I-IMP/125I-IMP metabolites and the effect of SKF-525A. Variations in CYP activity in vivo
were detected by administration of 123I-IMP and/or SKF-525A to mice. The liver and the kidney were thenexcised, homogenized and analyzed using HPLC.
Results: 125I-IMP was metabolized by MLH in the presence of NADPH, and the production of 125I-PIA wasinhibited by SKF-525A. SKF-525A did not greatly affect the biodistribution of 125I-IMP/ 125I-IMP metabolites
in vivo. Both 123I-IMP and 123I-PIA were detected in organs of control mice. However, 123I-PIA was not detectedin the livers or kidneys of mice treated with SKF-525A.
Conclusions: CYP activity in vivo was inhibited by SKF-525A treatment. Variations in CYP activity could be detectedin the blood, liver and kidney as changes in the peak area of 123I-PIA.
Advances in knowledge and implications for patient care: 123I-IMP metabolite analysis has the potential to provide
beneﬁcial information for prediction of the effect of medicines, in addition to its contribution to more accurate
rCBF diagnosis that reﬂects individual CYP activity.
© 2014 Elsevier Inc. All rights reserved.

1. Introduction
The ideal radiopharmaceutical is generally not metabolized anddoes not undergo a structural change in vivo. Radiopharmaceuticals
can be used for both tissue accumulation studies and/or for tissue
metabolic studies. For the former study, the ideal radiopharmaceutical

Disclosure/conﬂict of interest: The authors declare no conﬂict of interest.
⁎ Corresponding author at: School of Health Science, College of Medical, Pharmaceuticaland Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200942, Japan. Tel.: +81 76 265 2527; fax: +81 76 234 4366.
E-mail address: kei@mhs.mp.kanazawa-u.ac.jp (K. Kawai).
http://dx.doi.org/10.1016/j.nucmedbio.2014.08.015
0969-8051/© 2014 Elsevier Inc. All rights reserved.

is generally not metabolized and does not undergo a structural change
in vivo. For the latter study, the ideal radiopharmaceutical is only
metabolized after its accumulation in the target tissue. An importantfeature of nuclear medicine images is that these images reﬂect biological
functions. Radiopharmaceuticals that are administered to the bodyprovide information regarding the biological functions that are involvedin their accumulation, which vary according to the chemical structure of
the radiopharmaceutical. If the structure of the radiopharmaceuticalchanges before it goes into the tissues, then the behavior of this modiﬁed
radiopharmaceutical will differ from that of the administered parental
radiopharmaceutical. Thus, nuclear medicine images obtained withsuch a radiopharmaceutical would not accurately reﬂect biological

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K. Nishi et al. / Nuclear Medicine and Biology 42 (2015) 171–176

function. However, some radiopharmaceuticals are known to be metabolized in vivo and such radiopharmaceuticals can provide important biological data [1]. In particular, 123I-N-isopropyl-p-iodoamphetamine (123IIMP) is metabolized in stages and converted to 123I-p-iodoamphetamine(123I-PIA) [2].
Currently, 123I-IMP is widely used in clinical practice to examine
regional cerebral blood ﬂow (rCBF) [3,4]. The input function of 123I-IMP
is calculated based on octanol–water partition coefﬁcients because
the lipophilic nature of the 123I-IMP metabolites gradually decreases
as 123I-IMP is metabolized. However, 123I-IMP can potentially provide
not only rCBF data but also data regarding another important diagnostic
parameter; CYP activity. Our research group has identiﬁed CYP2C19
as the enzyme that metabolizes N-isopropyl-p-iodoamphetamine(cold-IMP) and has shown using high-performance liquid chromatography (HPLC) that CYP2C19 converts cold-IMP to p-iodoamphetamine(cold-PIA) in pooled microsomes from human liver (HLM) [5].
CYP2C19 is one of the cytochrome P450 enzymes that are involvedin the metabolism of medicines [6]. However, four phenotypes of the
CYP2C19 gene are expressed; extensive metabolizer (EM), intermediate
metabolizer (IM), poor metabolizer (PM), and ultra-rapid metabolizer(UM) [7]. In general, a PM has little ability to metabolize medicines,
and therefore is susceptible to side effects of such medicines. Thus, if
PM is the only, or the major CYP2C19 gene expressed, the subject issusceptible to side effects of normal doses of administered commonlyused medications.
It is therefore important to establish a method for measurement of
the medicine-metabolizing capacity of individuals. Our research groupfocused on analysis of the metabolites of 123I-IMP as a means of quantifying CYP2C19 activity in individuals. Since changes in 123I-PIA production may reﬂect variations in CYP2C19 activity, it should be possible todetermine CYP2C19 activity in individuals by quantitative analysis of
their 123I-PIA levels following 123I-IMP administration.
In the present study, we aimed to detect variations in CYP activity ina living organism by performing metabolic experiments in mice. These
data can be used as a basic study of 123I-IMP metabolite analysis forindividualized medicine.
2. Materials and methods
2.1. Materials
Reagent-grade chemicals (e.g. potassium dihydrogenphosphate,
dipotassium hydorogenphosphate, disodium edetate dihydrate,
60% perchloric acid, p-chlorobenzoic acid, and acetonitrile) werepurchased from Nacalai Tesque (Kyoto, Japan). Β-NADP + andglucose-6-phosphate dehydrogenase were obtained from Oriental
Yeast (Osaka, Japan). SKF-525A was obtained from Enzo Life Science(Zandhoven, Belgium). 125I-IMP and 123I-IMP were provided free by
Nihon Medi-Physics (Chiba, Japan).
2.2. Preparation of pooled homogenates of mouse liver (MLH)
Animal studies were approved by the Animal Care Committee atKanazawa University and were conducted in accordance with international standards for animal welfare and institutional guidelines.
Six male mice (ddY, 6 weeks old, Japan SLC, Tokyo, Japan) wereeuthanized with anesthesia using diethyl ether. The liver was thenremoved and weighed. After addition of Krebs–Ringer phosphate buffer(KRPB, pH 7.4) to the livers, the mixture was homogenized using an
ultrasonic homogenizer. The protein content was quantiﬁed using thebicinchoninic acid method [8].
2.3. Metabolism of 125I-IMP by MLH
NADPH-dependent metabolism of 125I-IMP (370 kBq) by the pooled
MLH samples was examined in an incubation mixture consisting of

100 mM sodium potassium phosphate buffer (pH 7.4), 50 μM EDTA
disodium salt, and 250, 500, 1000 or 2000 μg protein/10 μL pooled MLH
in a ﬁnal volume of 250 μL, in the presence or absence of an NADPHgenerating system (0.5 mM NADP +, 5 mM MgCl2, 5 mM glucose-6phosphate, and 1 U/mL glucose-6-phosphate dehydrogenase). A mixture
without MLH or NADPH was used as the control. The mixture wasincubated at 37 °C for 20, 40 or 60 min. The reaction was stopped byadding 50 μL perchloric acid following which p-chlorobenzoic acid
(20 μM, 30 μL) was added as an internal standard for ultraviolet (UV)
detection. Subsequently, the mixture was centrifuged for 5 min at
18000 g, and 100 μL supernatant was analyzed using HPLC equippedwith UV and gamma (γ)-ray detectors.
2.4. SKF-525A inhibition of 125I-IMP metabolism by MLH
SKF-525A was used as a selective CYP inhibitor [9,10]. Inhibitionof 125I-IMP (370 kBq) metabolism by pooled MLH was examined in
an incubation mixture consisting of 100 mM sodium potassium
phosphate buffer (pH 7.4), SKF-525A (ﬁnal concentration of 1, 5,
10 or 15 μM), 50 μM EDTA disodium salt and 1000 μg protein/10 μL
MLH in a ﬁnal volume of 250 μL, in the presence or absence of the
NADPH-generating system.
The mixture was incubated at 37 °C for 20 min. The reaction
was stopped by adding perchloric acid, following which 20 μM
p-chlorobenzoic acid was added as the internal standard. Subsequently,
these samples were centrifuged for 5 min at 18000 g, and 100 μL supernatant was analyzed using HPLC.
2.5. Biodistribution studies of 125I-IMP in mice
These studies were performed to evaluate the effects of SKF-525A on
the biodistribution of administered 125I-IMP. Male mice (ddY, 6 weeksold) were pretreated with SKF-525A in saline solution (1.25 μM,
100 μL) or with saline alone (100 μL), and, after 3 min, 125I-IMP(18.5 kBq, 100 μL) was administered via the tail vein. Mice were sacriﬁced
at 2, 5, 10, 15 and 20 min after injection of 125I-IMP by cervical dislocation
under deep ether anesthesia, and blood and organs of interest were
removed. Organs were weighed and their radioactivity was then determined using a scintillation counter. The ratio of radioactivity accumulated
per injected dose (ID) per organ weight was determined after decay
correction and is expressed as a percentage (%ID/g).
2.6. Metabolism of 123I-IMP and its inhibition by SKF-525A in miceMice were pretreated with or without SKF-525A (1.25 μM, 100 μL) in
saline solution, and, 3 min later, 123I-IMP (7.4 MBq, 100 μL) was administered via the tail vein. Fifteen minutes after 123I-IMP injection, mice weresacriﬁced by cervical dislocation under deep ether anesthesia. Blood
was collected and deproteinized by adding 50 μL perchloric acid. Subsequently, the mixture was centrifuged for 5 min at 18000 g, and 100 μL
supernatant was analyzed using HPLC. The liver and kidney were excised
and weighed. After addition of KRPB to the liver and kidney, the mixture
was homogenized using an ultrasonic homogenizer. The homogenates
were deproteinized by adding perchloric acid, centrifuged for 5 min at
18000 g, and 100 μL supernatant was analyzed using HPLC.
2.7. HPLC analysis of 125I- or 123I-IMP
125
I- or 123I-IMP and p-chlorobenzoic acid were analyzed using an
HPLC system consisting of a pump (model L-7100, Hitachi, Tokyo,
Japan), a UV detector (model SPD-10A, SHIMADZU, Tokyo, Japan) anda γ-ray detector (model RLC-701, Hitachi-Aloka Medical, Japan)
equipped with a 5C18 AR-II column (4.6 × 250 mm; 5 μm, Nacalai
Tesque). The mobile phase consisted of 60% (v/v) 20 mM potassium
phosphate buffer (pH 3.0) and 40% acetonitrile. Signal intensity was
expressed as voltage (V, or mV). The amount of 125I- or 123I-PIA was

K. Nishi et al. / Nuclear Medicine and Biology 42 (2015) 171–176

173

evaluated as the normalized peak area (%). The normalized peak area of
125
I- or 123I-PIA was deﬁned as follows: Ratio of the peak area of 125I- or
123
I-PIA to the total area of signiﬁcant peaks.
2.8. Selection of radioactive isotope (RI)
125

I-IMP was used instead of 123I-IMP for in vitro experiments andthe biodistribution study because of its longer half-life. However, todetect radioactivity in the in vivo CYP activity assay using the HPLC
system, it was necessary to have high levels of radioactivity in thehomogenates of the excised organs. Unfortunately, the concentrationof 125I-IMP in the homogenates was too low for detection by thisassay. Since the volume of 125I-IMP solution that would contain therequired radioactivity was too large to administer to mice, we therefore
used 123I-IMP for the in vivo CYP activity assay experiment.
2.9. Statistical analysis
All results represent the average of least three experiments andare expressed as the mean ± SD. Data were analyzed using Student's
t-test, and P b 0.05 was considered statistically signiﬁcant.
3. Results
125

I-IMP metabolism to 125I-PIA in MLH was analyzed using HPLC.
In in vitro experiments, the peaks of parental 125I-IMP and the metabolite 125I-PIA were detected in an incubation mixture containing
an NADPH-generating system as shown in Fig. 1A. In contrast, only
parental 125I-IMP was detected in a reaction mixture in the absence of
the NADPH-generating system (Fig. 1B). The normalized peak areaof 125I-PIA increased depending on the incubation time and the MLH
concentration (Fig. 2). At each incubation time, the normalized peakarea of 125I-PIA was maximal at 1000 μg protein/sample, and was
lower at 2000 μg protein/sample due to NADPH depletion.
In the metabolic inhibition experiment in vitro, 125I-PIA production
was reduced in a concentration-dependent manner following additionof the selective CYP inhibitor SKF-525A (Fig. 3). The normalized peakFig. 2. Relationship between MLH protein concentration and the normalized peak areaof 125I-PIA at each incubation time in vitro. MLH at the indicated protein concentration
was incubated with 125I-IMP for the indicated times in the presence of NADPH. Samples
were analyzed by HPLC and the normalized peak area of 125I-PIA was calculated. Error
bars represent the mean ± SD.

area of 125I-PIA was reduced by approximately 50% in the presence of
10 μM SKF-525A.
Tables 1A and 1B show the in vivo biodistribution of 125I ( 125I-IMP
plus 125I-PIA) in each mouse organ as a percentage of the injection
dose/g tissue (%ID/g) or/mL blood (%ID/mL) in the absence (1A) or
presence (1B) of SKF-525A. In both the control and the SKF-525A
group, 125I (125I-IMP plus 125I-PIA) showed higher accumulation in thekidney compared to other organs except for the lung, and the level ofradioactivity in the blood was very low. In addition, no radioactivity
was detected in the urine. Fig. 4 shows the in vivo data for blood, liverand kidney from Tables 1A and 1B in graph form and compares
the effect of SKF-525A on accumulated radioactivity. There was no
signiﬁcant difference in the radioactive blood levels (A), or in the accumulated radioactivity levels in the liver (B) between the groups withor without SKF-525A. However, in the kidney (C), the accumulated
level of 125I-IMP plus 125I-PIA at 5 min was higher in the SKF-525A
group than in the control group (P b 0.05). These results indicate slightly
faster distribution of 125I-IMP plus 125I-PIA to the kidney following
co-administration of SKF-525A.
In HPLC analysis of the blood of mice administered 123I-IMP, peaks ofparental 123I-IMP and the metabolite 123I-PIA were detected in both thecontrol and the SKF-525A groups. However, the normalized peak areaof 123I-PIA was markedly and signiﬁcantly reduced compared with thecontrol group due to the SKF-525A loading. Moreover, whereas peaksof both 123I-IMP and 123I-PIA were detected in organ homogenates
prepared from control mice, only a peak of parental 123I-IMP was
detected in organ homogenates prepared from mice loaded with
SKF-525A (Table 2).
4. Discussion
Considerable individual differences exist in the pharmacokinetics ofhumans. In most cases, the differences result from individual differences

Fig. 1. Chromatograms of in vitro metabolite analyses. MLH (1000 μg protein/sample) was
incubated in the presence (A) or absence (B) of NADPH for 60 min and then analyzed by
HPLC. The peaks of parental 125I-IMP and of 125I-PIA are shown. 125I-PIA was only detectedin the presence of NADPH.

Fig. 3. Effect of SKF-525A concentration on 125I-PIA generation in vitro. MLH (1000 μg
protein/sample) was incubated with 125I-IMP for 20 min in the presence of the indicatedconcentration of SKF-525A. The normalized peak area of 125I-PIA was determined using
HPLC. Error bars represent the mean ± SD. The normalized peak area of 125I-PIA decreased
with increasing SKF-525A concentration.

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K. Nishi et al. / Nuclear Medicine and Biology 42 (2015) 171–176

Table 1A
Biodistribution of 125I-IMP and its metabolites in mice not pretreated with SKF-525A.
Organ

Blood
Brain
Lung
Heart
Liver
Pancreas
SpleenStomachKidneyIntestine
MuscleUrine

Time after 125I-IMP injection
2 min

5 min

10 min15 min20 min0.67 ± 0.07
5.46 ± 0.48
21.79 ± 5.60
5.51 ± 0.85
2.68 ± 0.29
4.40 ± 1.61
2.49 ± 0.51
2.70 ± 0.48
7.92 ± 1.08
1.98 ± 0.15
2.77 ± 0.54
N.D.

0.64 ± 0.16
5.36 ± 0.96
16.82 ± 5.14
4.06 ± 0.88
3.44 ± 0.79
6.85 ± 1.58
3.85 ± 0.63
3.42 ± 0.86
9.43 ± 1.25
2.13 ± 0.40
2.27 ± 0.19
N.D.

0.74 ± 0.11
7.29 ± 1.96
17.70 ± 4.65
4.10 ± 0.98
4.97 ± 0.43
10.35 ± 2.22
8.15 ± 0.87
7.13 ± 2.10
11.87 ± 1.92
3.09 ± 0.38
2.19 ± 0.13
N.D.

0.74 ± 0.12
6.42 ± 0.09
13.82 ± 1.26
3.93 ± 0.68
4.67 ± 0.08
11.16 ± 0.90
4.54 ± 1.62
7.05 ± 0.03
12.04 ± 0.54
2.75 ± 0.04
1.78 ± 0.15
N.D.

0.76 ± 0.04
6.57 ± 0.26
13.14 ± 1.79
3.33 ± 0.02
4.79 ± 0.19
10.63 ± 0.24
6.66 ± 0.16
8.35 ± 1.88
10.33 ± 0.83
2.80 ± 0.19
1.90 ± 0.15
N.D.

%ID/g was calculated with the following formula: 100 × (cpm of organ/injected dose)/organ weight. N.D.: Not detected.

in the activity of drug-metabolizing enzymes as typiﬁed by CYP. Genetic
polymorphisms are cited as the main factors responsible for individual
differences in the activity of drug-metabolizing enzymes [11,12].
Individuals with very low or very high metabolic capacities exist due
to genetic polymorphisms in CYP, and are described as PM and EM
respectively [13,14]. Currently, such individual differences at the genetic
level are distinguished by genetic analysis. However, genetic analysis
cannot determine individual metabolic capacity at a particular point oftime, which involves a variety of factors such as concomitant ingestionof different medicines, alcohol intake and deterioration in liver function
due to aging. On the other hand, our method based on metabolite
analysis can measure an individual's metabolic capability thatreﬂects all of the factors described above. As previously indicated,to date 123I-IMP has only been used to examine rCBF. However, basedon our data, 123I-IMP will be able to provide diagnostic information
regarding metabolic function. On a relevant note, it will also be possible
to accurately calculate input function by measuring the amount ofan 123I-IMP metabolite because the ratio of lipophilic metabolites that
contaminate the octanol fraction will change according to the CYP activityof the individual patient. Thus, the present study provides an importantbasis for accurate assessment of input function and for establishmentof an index that can be used to determine the dose of medicine to beprescribed for individuals. In brief, competition with 123I-IMP can beused for dose determination of medicines that are non-radioactive
and are substrates of CYP2C19, such as imipramine, clomipramine,omeprazole and cimetidine. These non-radioactive medicines inhibit
metabolism of 123I-IMP in a similar manner to SKF-525A.
In our previous study with HLM, cold-IMP was metabolized by
CYP2C19 to cold-PIA [5]. However, IMP-metabolizing enzymes in mice

have not been identiﬁed, and important species differences are present
between humans and mice. In addition, the number of molecules of125
I- or 123I-IMP that are accumulated in HLM or in organs afterin vivo administration is very low. For these reasons, it was necessary
to detect 125I- or 123I-IMP, as well as cold-IMP, using an analyticalmethod such as HPLC.
CYPs are drug-metabolizing enzymes that act when NADPH ispresent as an energy source [15]. In in vitro experiments, 125I-IMP
and 125I-PIA were detected with high sensitivity using HPLC, and we
conﬁrmed that 125I-IMP was metabolized and converted to 125I-PIA
only in the presence of NADPH. In addition, the normalized area of
the 125I-PIA peak increased in a manner that was dependent on theprotein concentration of MLH and on the incubation time (Fig. 2). We
therefore concluded that the metabolism of 125I-IMP was due to an
enzymatic reaction. Based on these results, the protein concentrationand incubation time were set at 1000 μg/sample and 20 min respectively,
because a sufﬁcient normalized peak area was obtained under these
conditions that permitted evaluation of changes in the amount of 125I-PIA.
The aim of the in vitro experiments of metabolic inhibition was to
conﬁrm that IMP is metabolized by mouse CYP. Since it is unknown
which CYP family metabolizes 125I-IMP in mice, we used SKF-525A as a
CYP inhibitor, because SKF-525A inhibits the function of CYP regardlessof the family or subfamily classiﬁcation of CYP. The normalized areaof the 125I-PIA peak was reduced in an SKF-525A concentrationdependent manner (Fig. 3). Based on these results, we inferredthat 125I-IMP was metabolized by CYP in mice as well as in humans.
In the biodistribution study, 125I-PIA cannot be distinguishedfrom 125I-IMP, because they both contain the same radionuclide; 125I.
Therefore this study evaluated the overall biodistribution of 125I-IMP

Table 1B
Biodistribution of 125I-IMP and its metabolites in mice pretreated with SKF-525A.
Organ

Blood
Brain
Lung
Heart
Liver
Pancreas
SpleenStomachKidneyIntestine
MuscleUrine

Time after 125I-IMP injection
2 min

5 min

10 min15 min20 min0.87 ± 0.36
5.95 ± 2.22
17.88 ± 7.35
5.32 ± 2.28
3.35 ± 0.57
6.89 ± 3.24
4.60 ± 1.84
3.68 ± 1.66
10.11 ± 2.46
2.09 ± 0.24
2.27 ± 0.67
N.D.

0.85 ± 0.03
6.83 ± 0.21
16.11 ± 2.11
5.29 ± 0.67
4.53 ± 0.10
8.46 ± 1.37
5.83 ± 0.73
4.81 ± 1.32
11.90 ± 0.12
2.49 ± 0.15
2.83 ± 0.32
N.D.

0.91 ± 0.18
7.52 ± 0.98
18.91 ± 4.73
5.44 ± 0.17
5.85 ± 1.40
9.87 ± 0.59
6.64 ± 0.75
7.67 ± 0.69
13.46 ± 1.52
3.24 ± 0.34
2.54 ± 0.25
N.D.

0.80 ± 0.29
6.96 ± 2.27
17.52 ± 5.53
4.38 ± 1.54
5.08 ± 1.25
7.63 ± 3.71
6.80 ± 0.94
9.04 ± 3.63
12.01 ± 1.15
2.72 ± 0.71
1.99 ± 0.89
N.D.

0.81 ± 0.12
7.31 ± 0.59
16.32 ± 3.47
4.42 ± 0.30
4.76 ± 0.12
9.87 ± 0.63
7.65 ± 0.52
9.24 ± 3.38
10.89 ± 1.10
2.52 ± 0.22
2.24 ± 0.19
N.D.

%ID/g was calculated with the following formula: 100 × (cpm of organ/injected dose)/organ weight. N.D.: Not detected.

K. Nishi et al. / Nuclear Medicine and Biology 42 (2015) 171–176

Fig. 4. Graphic analysis of the in vivo effect of SKF-525A on the accumulation of 125I-IMP
and its metabolites in speciﬁc organs. Radioactive (125I) levels in the blood (A), liver(B) and kidney (C) of mice pretreated with SKF-525A or saline for 3 min prior to injectionof 125I-IMP, as indicated in Tables 1A and 1B, are shown in graph form. ID; injected
dose. Error bars represent the mean ± SD. *: P b 0.05 compared with control group. Little
difference was noted between the control and SKF-525A groups.

125

including
I-IMP metabolites. This biodistribution study indicated asigniﬁcant difference in the accumulation of 125I-IMP/125I-IMP metabolites in the kidney at 5 min between the control and the SKF-525A
group. However, no signiﬁcant difference was found in the accumulationof 125I-IMP at other times or in other tissues between the two groups.
Thus, we observed few signiﬁcant alterations in the pharmacokineticsof 125I-IMP due to preloading of SKF-525A. In both the control andSKF-525A groups, radioactivity in organs had almost reached equilibrium
15 min after administration of 125I-IMP, indicating that tissue migrationof 125I-IMP had reached a steady state at this time point. As shown in
Table 2, following preloading of SKF-525A, the peak of 123I-PIA disappeared in the liver and kidney and the normalized peak area of 123I-PIA
was signiﬁcantly reduced in the blood. We considered that these
decreases in 123I-PIA levels were caused by decreased CYP activity, andnot by administration of SKF-525A because the radioactivity in otherorgans was not signiﬁcantly altered (Tables 1A and 1B and Fig. 4).

Table 2
Effect of SKF-525A on 123I-PIA levels in organs of 123I-IMP-treated mice.
Organ

Control group

SKF-525A group

Blood
KidneyLiver

25.78 ± 1.74
14.38 ± 7.31
25.17 ± 7.61

15.13 ± 1.10
N.D.
N.D.

The normalized area of the 123I-PIA peak in HPLC analysis is shown. N.D.: Not detectable.

175

In general, careful consideration is necessary in discussion of themetabolism of medicines administered at different concentrations,because it is known that the velocity of metabolic enzymatic reactions
depends on the concentration of the substrate, and, furthermore, thatmetabolic enzymes other than the CYP family are present. However,when the focus is on main and important one enzyme and its labeled
substrate, it is possible to discuss the metabolism of the labeled
substrate that is administered at tracer concentrations based on thepremise that the species and function of the enzymes that metabolize
this substrate do not change in the presence of a tracer concentration of
the substrate. An example of such a study is the microdose study reported
by Tozuka et al [16]. The fact that there is no pharmacological change inin vivo conditions due to the presence of a tracer concentration of the substrate is an advantage in this kind of study. In the present study, 123I-IMP
administration does not exert a pharmacological effect because 123I-IMP
is present in trace amounts. However, the administered 123I-IMP would
accurately reﬂect individual metabolic function.
Although the present experiments were performed using mice, we
consider that the metabolic rate of 123I-IMP will similarly ﬂuctuate
depending on CYP activity in humans.
In humans, a medicine that is a substrate for CYP2C19 would behave
as a CYP2C19 inhibitor instead of SKF-525A. Subsequent 123I-IMP
metabolite analysis would then indicate variation in CYP2C19 activity
due to ingestion of the medicine.
In addition, the only radiopharmaceutical that can currently be used
for CYP activity-measurement methods that are based on metabolicchanges is 123I-IMP. This is because radiopharmaceuticals used in thismethod must meet the following conditions: a metabolizing enzyme
must have been identiﬁed, its metabolites must also be radioactive,and detection and separation from the parental substance must beeasy. This method of estimation of CYP activity using 123I-IMP metabolite analysis has the potential to contribute to more accurate functional
diagnosis that reﬂects individual metabolic function, and to the establishment of an index for determination of the correct dose of medicine
to be administered in humans. Therefore, this method will lead to theestablishment of individualized medicine.
5. Conclusion
Our aim was to detect CYP activity in vivo by quantitative analysisof 123I-IMP metabolites. In the present study, we have demonstrated
the following: First, 123I-IMP and 123I-PIA in HMLC are detectableusing HPLC. Second, the normalized peak area of 123I-PIA decreased
due to reduction of CYP activity caused by SKF-525A treatment. Finally,variations in CYP activity in vivo were detected in the blood, liver,and kidney of mice as changes in the normalized area of the 123I-PIA
HPLC peak.
For clinical application, it is necessary that 123I-PIA can be detected insamples such as blood or urine that can be collected in a minimallyinvasive manner, instead of in liver or kidney. In addition, it will benecessary to ﬁnd a simpler method of analysis, such as thin-layer
chromatography, instead of HPLC. However, we conﬁdently conclude
that 123I-IMP metabolite analysis can be used to detect variations in
CYP activity in vivo. Consequently, metabolite analysis of 123I-IMP canpotentially be used in a clinical setting as a method for rCBF analysis
that reﬂects metabolic function and estimates the metabolic capacityof patients.
Acknowledgments
The authors are grateful to Nihon Medi-Physics Co., Ltd., forgenerously providing 125I- and 123I-IMP. We also thank Ayana Ishiguro,
Tatsuya Sano, Takafumi Tsujiuchi, Masatoshi Sakashita and Takuya
Ohmichi for their assistance with the biodistribution study of 125Ior 123I-IMP.

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References[1] Cumming P, Yokoi F, Chen A, Deep P, Dagher A, Reutens D, et al. Pharmacokinetics ofradiotracers in human plasma during positron emission tomography. Synapse 1999;
34:124–34.
[2] Baldwin RM, Wu JL. In vivo chemistry of iofetamine HCl iodine-123 (IMP). J Nucl
Med 1988;29:122–4.
[3] Cohen MB, Graham LS, Yamada LS. [123I]iodoamphetamine SPECT imaging. Int J
Radiat Appl Instrum A 1986;37:749–63.
[4] Mimura H, Sone T, Takahashi Y, Yoshioka K, Murase K, Matsuda H, et al. Measurementof regional cerebral blood ﬂow with 123I-IMP using one-point venous blood sampling
and causality analysis: evaluation by comparison with conventional continuousarterial blood sampling. Ann Nucl Med 2006;20:589–95.
[5] Fujita K, Sugiyama M, Akiyama Y, Hioki K, Kunishima M, Nishi K, et al. N-Isopropylp-iodoamphetamine hydrochloride is predominantly metabolized by CYP2C19.
Drug Metab Dispos 2012;40:843–6.
[6] Daly AK. Pharmacogenetics of the major polymorphic metabolizing enzymes.
Fundam Clin Pharmacol 2003;17:27–41.
[7] Saeed LH, Mayet AY. Genotype-phenotype analysis of CYP2C19 in healthy Saudi
individuals and its potential clinical implication in drug therapy. Int J Med Sci
2013;10:1497–502.
[8] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al.
Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76–85.

[9] Kauser K, Clark JE, Masters BS, de Montellano PR Ortiz, Ma YH, Harder DR, et al.
Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renalarcuate arteries. Circ Res 1991;68:1154–63.
[10] Nunoya K, Yokoi Y, Kimura K, Kodama T, Funayama M, Inoue K, et al. (+)-cis-3,5dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502) as a novel
substrate for cytochrome P450 2A6 in human liver microsomes. J Pharmacol Exp
Ther 1996;277:768–74.
[11] van der Weide J, Steijns LS. Cytochrome P450 enzyme system: genetic polymorphisms and impact on clinical pharmacology. Ann Clin Biochem 1999;36(Pt 6):
722–9.
[12] Singh D, Kashyap A, Pandey RV, Saini KS. Novel advances in cytochrome P450
research. Drug Discov Today 2011;16:793–9.
[13] Alonso-Navarro H, Jimenez-Jimenez FJ, Garcia-Agundez JA. The role of CYP2C19
polymorphism in the development of adverse effects to drugs and the risk for
diseases. Med Clin 2006;126:697–706.
[14] Wijnen PA, Op den Buijsch RA, Drent M, Kuijpers PM, Neef C, Bast A, et al. Review
article: the prevalence and clinical relevance of cytochrome P450 polymorphisms.
Aliment Pharmacol Ther 2007;26(Suppl. 2):211–9.
[15] Reed JR, Backes WL. Formation of P450. P450 complexes and their effect on P450
function. Pharmacol Ther 2012;133:299–310.
[16] Tozuka Z, Kusuhara H, Nozawa K, Hamabe Y, Ikushima I, Ikeda T, et al. Microdose
study of 14C-acetaminophen with accelerator mass spectrometry to examine pharmacokinetics of parent drug and metabolites in healthy subjects. Clin Pharmacol
Ther 2010;88:824–30.